White 384 well plate, by PerkinElmer - Product details

1

Europium-labeled Peptide Assay for DsbA

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Assays were run on a Synergy H1 Multi-Mode plate reader (BioTek, USA) with excitation at 340 nm and emission at 615 nm. For time-resolved fluorescence, a 100 µs delay before reading and a 200 µs reading time were employed. The assay was performed in a white 384-well plate (PerkinElmer). A 25 µl solution consisting of 3.2 µM EcDsbA and 2 mM GSSG (positive control) or of 3 µM FL α-DsbA2 or α-DsbA2ΔN and 2 mM GSSG in 50 mM MES, 50 mM NaCl, 2 mM EDTA pH 5.5 was added to the wells. The assay was initiated by the addition of 25 µl 16 µM peptide (in 50 mM MES, 50 mM NaCl, 2 mM EDTA pH 5.5) to each well. The peptide substrate was CQQGFDGTQNSCK, with europium bound to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group amide-coupled to the N-terminus, and a methylcoumarin amide-coupled to the ∊-amino group of the C-terminal lysine (AnaSpec, USA). Reconstitution of the peptide substrate solution is described in Walden et al. (2012 ▸ ). Measurements were carried out in triplicate using three different protein batches and the data shown are the mean ± SD error from these three measurements.

Walden P.M., Whitten A.E., Premkumar L., Halili M.A., Heras B., King G.J, & Martin J.L. (2019). The atypical thiol–disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase. Acta Crystallographica. Section D, Structural Biology, 75(Pt 3), 283-295.

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2

Quantitative K-Ras4B-RAF Kinase Interaction

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K-Ras4B:RAF kinase interaction assays were performed as previously described (16 (link)). Purified RAF-1 RBD was labeled with maleimide PEG biotin (Pierce) following the manufacturer’s recommended protocol. Purified flag-tagged K-Ras4B (1 mg/mL) were loaded with GMPPNP (Sigma-Aldrich) by incubating for 2 hours at 25°C with a 50-fold excess of nucleotide in the presence of alkaline phosphatase (Thermo-Fisher). RAF–RBD–biotin was diluted to a final concentration of 40 nM and Flag-K-Ras4B to 10 nM in assay buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 5% glycerol, 0.5% BSA) and added to individual wells of a low-volume white 384-well plate (PerkinElmer). Complexes were disrupted by addition of a dilution series (2,000 nM to 0.5 nM) of each mutant K-Ras4B protein. The assay was developed by addition of streptavidin donor and anti-flag acceptor AlphaScreen beads (10 μg/mL). Alpha signal was measured after overnight incubation at 4°C. All readings were performed in triplicate.

Poulin E.J., Bera A.K., Lu J., Lin Y.J., Strasser S.D., Paulo J.A., Huang T.Q., Morales C., Yan W., Cook J., Nowak J.A., Brubaker D.K., Joughin B.A., Johnson C.W., DeStefanis R.A., Ghazi P.C., Gondi S., Wales T.E., Iacob R.E., Bogdanova L., Gierut J.J., Li Y., Engen J.R., Perez-Mancera P.A., Braun B.S., Gygi S.P., Lauffenburger D.A., Westover K.D, & Haigis K.M. (2019). Tissue-specific oncogenic activity of K-RasA146T. Cancer discovery, 9(6), 738-755.

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3

KRAS:Raf Binding Affinity Assay

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An AlphaScreen^® (Perkin Elmer) assay was used to measure the affinity of KRAS^P34R:nucleotide complexes for the Ras Binding Domain (RBD) of the Raf protein kinase. Purified RAF kinase RBD was labeled with maleimide PEG biotin (Pierce) following the manufacturer's protocol. Purified flag-tagged KRAS (1 mg/ml) and KRAS^P34R mutant were loaded with GMPPNP (Sigma-Aldrich) by incubating for 2 hr at 25° C with a 50-fold excess of nucleotide in the presence of alkaline phosphatase (ThermoFisher). RAF–RBD–biotin was diluted to a final concentration of 40 nmol/L and Flag-KRAS to 10 nmol/L in assay buffer (20 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 1 mmol/L MgCl₂, 5% glycerol, 0.5% BSA) and added to individual wells of a low volume white 384-well plate (PerkinElmer). Complexes were disrupted by addition of untagged WT KRAS or KRAS^P34R mutant preloaded with GMPPNP over a range of concentrations. The assay was developed by addition of streptavidin donor and anti-flag acceptor AlphaScreen beads (10 mg/ml). Alpha signal was measured after overnight incubation at 4°C.

Bera A.K., Lu J., Lu C., Li L., Gondi S., Yan W., Nelson A., Zhang G, & Westover K.D. (2020). GTP hydrolysis is modulated by Arg34 in the RASopathy-associated KRASP34R. Birth defects research, 112(10), 708-717.

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4

PI3K Kinase Activity Assay

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Compound YXY-4F was dissolved in 100% dimethylsulfoxide (DMSO) at 10 mM. The solution was diluted to the desired concentrations immediately before each experiment. The kinase activities of the purified PI3Ks were determined with the PI3K HTRF Assay (Millipore) according to the manufacturer’s protocol. Briefly, the EC₈₀ concentration of each enzyme containing 10 μM PIP2 was incubated in assay buffer on a white 384-well plate (Perkin Elmer). After incubation for 30 min at room temperature, the reaction was initiated by addition of ATP and terminated by addition of the stop solution and detection mix. The final concentration of ATP was 5 μM for p110α. The plate was then sealed and incubated overnight at room temperature. The intensity of the light emission was measured by an EnVision Multilabel Reader (PerkinElmer) in TR-FRET mode (excitation at 320 nm and emission at 665 nm) as previously described^{42 (link)}. The IC₅₀ values^{43 (link)} were calculated by fitting the data to a logistic curve using the GraphPad Prism 6 software.

Yang X., Zhang X., Huang M., Song K., Li X., Huang M., Meng L, & Zhang J. (2017). New Insights into PI3K Inhibitor Design using X-ray Structures of PI3Kα Complexed with a Potent Lead Compound. Scientific Reports, 7, 14572.

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5

Optimizing PPAR-γ Transcriptional Assay

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HEK293T cells (ATCC; cat# CRL-3216) were cotransfected in batch by adding 4.5 μg human GAL4-PPARγ-Hinge-LBD, with 4.5 μg 5X multimerized UAS-luciferase reporter and 27 μL X-treme Gene 9 transfection reagent in serum-free Opti-mem reduced serum media (Gibco). After 18-h incubation at 37 °C in a 5% CO₂ incubator, transfected cells were plated in quadruplicate in white 384-well plates (Perkin Elmer) at a density of 10,000 cells per well. After replating, cells were treated with either DMSO only or the indicated compounds in increasing doses from 2 pM–10 μM. After 18-h incubation, treated cells were developed with Brite Lite Plus (Perkin Elmer) and read in 384-well Luminescence Perkin Elmer EnVision Multilabel plate reader. Graphs were plotted as fold change of treated cells over DMSO-treated control cells.

Frkic R.L., Chua B.S., Shin Y., Pascal B.D., Novick S.J., Kamenecka T.M., Griffin P.R, & Bruning J.B. (2018). Structural and Dynamic Elucidation of a Non-acid PPARγ Partial Agonist: SR1988. Nuclear receptor research, 5, 101350.

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6

PPARγ2 Transcriptional Activation Assay

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HEK293T cells (ATCC; cat# CRL-3216) were cotransfected in batch by adding 4.5 µg full-length human PPARγ2-pCMV-AN-DDK or full-length human mutant PPARγ2-F282A-pCMV-AN-DDK, with 4.5 µg 3x multimerized PPRE-luciferase reporter and 27 µl X-treme Gene 9 transfection reagent in serum-free Opti-mem reduced serum media (Gibco). After 18-h incubation at 37 °C in a 5% CO₂ incubator, transfected cells were plated in quadruplicate in white 384-well plates (Perkin Elmer) at a density of 10,000 cells per well. After replating, cells were treated with either DMSO only or the indicated compounds in increasing doses from 2 pM–10 µM. After 18-h incubation, treated cells were developed with Brite Lite Plus (Perkin Elmer) and read in 384-well Luminescence Perkin Elmer EnVision Multilabel plate reader. Graphs were plotted as fold change of treated cells over DMSO-treated control cells.

Marciano D.P., Kuruvilla D.S., Boregowda S.V., Asteian A., Hughes T.S., Garcia-Ordonez R., Corzo C.A., Khan T.M., Novick S.J., Park H., Kojetin D.J., Phinney D.G., Bruning J.B., Kamenecka T.M, & Griffin P.R. (2015). Pharmacological Repression of PPARγ Promotes Osteogenesis. Nature communications, 6, 7443.

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7

Optimizing PPAR-γ Transcriptional Assay

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HEK293T cells (ATCC; cat# CRL-3216) were cotransfected in batch by adding 4.5 μg human GAL4-PPARγ-Hinge-LBD, with 4.5 μg 5X multimerized UAS-luciferase reporter and 27 μL X-treme Gene 9 transfection reagent in serum-free Opti-mem reduced serum media (Gibco). After 18-h incubation at 37 °C in a 5% CO₂ incubator, transfected cells were plated in quadruplicate in white 384-well plates (Perkin Elmer) at a density of 10,000 cells per well. After replating, cells were treated with either DMSO only or the indicated compounds in increasing doses from 2 pM–10 μM. After 18-h incubation, treated cells were developed with Brite Lite Plus (Perkin Elmer) and read in 384-well Luminescence Perkin Elmer EnVision Multilabel plate reader. Graphs were plotted as fold change of treated cells over DMSO-treated control cells.

Frkic R.L., Chua B.S., Shin Y., Pascal B.D., Novick S.J., Kamenecka T.M., Griffin P.R, & Bruning J.B. (2018). Structural and Dynamic Elucidation of a Non-acid PPARγ Partial Agonist: SR1988. Nuclear receptor research, 5, 101350.

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8

GAL4-PPARγ Transactivation Assay

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HEK 293T cells (ATCC; cat. no. CRL-3216) were cotransfected in batch by adding 4.5 μg of human GAL4-PPARγ-Hinge-LBD with 4.5 μg of 5× multimerized UASluciferase reporter and 27 μL of X-treme Gene 9 transfection reagent in serum-free Opti-MEM reduced serum media (Gibco). Cells cotransfected with Gal4-pBind vector and UAS-LUC were used as a nonreceptor control. After 18 h of incubation at 37 °C in a 5% CO₂ incubator, transfected cells were plated in quadruplicate in white 384- well plates (PerkinElmer) at a density of 10 000 cells per well. After replating, cells were treated with either DMSO only or the indicated compounds in increasing doses from 2 pM to 10 μM. After an 18 h incubation, treated cells were developed with Brite Lite Plus (PerkinElmer) and read in 384-well Luminescence PerkinElmer EnVision Multilabel plate reader. Graphs were plotted as fold change of treated cells over DMSO-treated control cells. Experiments were performed in triplicate, and statistical analysis including the incorporation of error bars was carried out in GraphPad Prism.

van Marrewijk L.M., Polyak S.W., Hijnen M., Kuruvilla D., Chang M.R., Shin Y., Kamenecka T.M., Griffin P.R, & Bruning J.B. (2015). SR2067 Reveals a Unique Kinetic and Structural Signature for PPARγ Partial Agonism. ACS chemical biology, 11(1), 273-283.

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9

PPARγ2 Transcriptional Activation Assay

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HEK293T cells (ATCC; cat# CRL-3216) were cotransfected in batch by adding 4.5 µg full-length human PPARγ2-pCMV-AN-DDK or full-length human mutant PPARγ2-F282A-pCMV-AN-DDK, with 4.5 µg 3x multimerized PPRE-luciferase reporter and 27 µl X-treme Gene 9 transfection reagent in serum-free Opti-mem reduced serum media (Gibco). After 18-h incubation at 37 °C in a 5% CO₂ incubator, transfected cells were plated in quadruplicate in white 384-well plates (Perkin Elmer) at a density of 10,000 cells per well. After replating, cells were treated with either DMSO only or the indicated compounds in increasing doses from 2 pM–10 µM. After 18-h incubation, treated cells were developed with Brite Lite Plus (Perkin Elmer) and read in 384-well Luminescence Perkin Elmer EnVision Multilabel plate reader. Graphs were plotted as fold change of treated cells over DMSO-treated control cells.

Marciano D.P., Kuruvilla D.S., Boregowda S.V., Asteian A., Hughes T.S., Garcia-Ordonez R., Corzo C.A., Khan T.M., Novick S.J., Park H., Kojetin D.J., Phinney D.G., Bruning J.B., Kamenecka T.M, & Griffin P.R. (2015). Pharmacological Repression of PPARγ Promotes Osteogenesis. Nature communications, 6, 7443.

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10

Protein-Protein Interaction Assay with AlphaScreen

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AlphaScreen Technology was used to assess the interactions between GST-E6, GST-caspase 8, His-E6AP, and His-caspase 8. Binding assays were performed in white 384-well plates (Perkin-Elmer) in a total volume of 25 μl as previously described.^{33 (link)} Briefly, 5 μl (50 ng) of GST-E6 and 5 μl (87.5 ng) of His-caspase 8 were included in each reaction mixture with 5 μl blocking buffer (0.5 mg BSA, 0.5% Tween 20 in PBS) in the absence or presence of 10 μM of each test chemical. Members of the library were present at 10 μM in DMSO. After a 1-h incubation of the mixture at room temperature, 5 μl donor beads and 5 μl acceptor beads (Perkin-Elmer) were added to each well according to the manufacturer's protocol. The mixture was incubated in the dark at room temperature overnight, and the emitted signal was detected using the Envision Multilabel plate reader (Perkin-Elmer). In the presence of test chemicals, the binding affinity was calculated as a percentage of the binding in the presence of carrier only (DMSO).

Yuan C.H., Filippova M., Krstenansky J.L, & Duerksen-Hughes P.J. (2016). Flavonol and imidazole derivatives block HPV16 E6 activities and reactivate apoptotic pathways in HPV+ cells. Cell Death & Disease, 7(1), 2060-.

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White 384 well plate

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25 protocols using white 384 well plate

Europium-labeled Peptide Assay for DsbA

Quantitative K-Ras4B-RAF Kinase Interaction

KRAS:Raf Binding Affinity Assay

PI3K Kinase Activity Assay

Optimizing PPAR-γ Transcriptional Assay

PPARγ2 Transcriptional Activation Assay

Optimizing PPAR-γ Transcriptional Assay

GAL4-PPARγ Transactivation Assay

PPARγ2 Transcriptional Activation Assay

Protein-Protein Interaction Assay with AlphaScreen

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